Probiotic bacteria such as Bacillus subtilis and Bacillus licheniformis are used in the animal feed industry as supplement to the diet. Their usage is related to the ability of bacillus to replace or reduce the use of antibiotics, which are used as growth promoters in the animal feed industry.
Christian Hansen A/S, Denmark commercializes an example of such a probiotic growth-promoting product under the trade name GalliPro® (deposited as DSM 17231). GalliPro® is a Bacillus subtilis spore cell composition.
Besides the suggested mode of actions (e.g. immune modulation, gut flora modifier) probiotic bacillus are able to produce many beneficial components, such as enzymes, which are excreted in the gastro intestinal tract (GIT) when used as animal feed supplement. Enzymes such as phytase are excreted and improve the digestion and better uptake of animal feed (higher digestibility). The diet (feed) is mostly composed of plant origin such as grains, corn, soybean, soy oil and amino acids. Overall these effects contribute to the production of cost effective animal products. One of the widely used enzymes in the animal feed industry is phytase. Phytase is applied for improving the digestibility of phosphorous in animal diets. Phytate is the predominant form of phosphorus in cereal grains, oilseeds and legumes. However, monogastric animals, such as pigs, poultry and fish, utilize this source of phosphate poorly because they lack the requisite gastrointestinal tract enzyme for release of the phosphate from the organic complex of phytate. Consequently, a large proportion of phytate in the feed consumed is passed through the GI-tract and excreted in the manure. In soil and water environments the catalyzed release of phosphate occurs, and phytate in manure poses a serious phosphorus pollution problem contributing to the eutrophication of surface waters. In addition, producers have to use expensive supplementary feed phosphorus to meet animals' dietary requirements. Further, phytate has anti-nutritive properties including formation of complexes with proteins and divalent cat ions, thus reducing their bioavailability.
It has been well documented that phytase supplementation improves phosphate use in monogastric production animals, and has a positive effect on the bioavailability of minerals.
Bacillus spores can pass the acidic gastric barrier and germinate and outgrow within the gastrointestinal (GIT) of the animals. This has great advantages, since when ingested they can excrete numerous types of beneficial components, e.g. bacteriocins and also excrete useful enzymes such as phytase. Moreover, the bacillus spores are thermostabile during a feed pelletizing process and are thereby an excellent delivery system to get both bacteriocins and enzymes into the GIT.
In the survival and proliferation process of bacillus in GIT, the role of bile is important. Bile is produced in the liver and stored in the gallbladder. Bile contains water, lecithin, bilirubin and biliverdin and bile salts.
It is known from the literature that bile has some negative influences on the survival and germination and outgrowth of bacillus spore cells to vegetative cells in the GIT of animals. Therefore research is ongoing to find probiotic bile resistant Bacillus strains.
The article (Antonie Van Leeuwenhoek. 2006 August; 90(2): 139-46. Epub 2006 Jul. 4) describes isolation of a number of Bacillus samples/cell directly from the intestine of chickens. The isolated bacillus cells were tested for probiotic activity. The six bacilli with highest probiotic activity were testes for bile salt resistance and it was found that a specific highly probiotic bacillus has a relatively high level of bile salt resistance.
In this article there is no special focus on any time periods for the testing of bile resistance. In the experimental part the bacillus spore cells are simply tested for resistance after 5 days of presence in bile salt (see paragraph “Simulated small intestinal fluid tolerance test” on page 141).
US2003/0124104A describes that probiotic conventional bacillus endospores are sensitive to low concentration of bile salts, i.e. spore germination and/or rehydration is inhibited by the presence of even low concentrations of bile salts. This is contrary to other bacteria such as enteric pathogens, such as E. coli or S. aureus (see section [0014] to [0015]). In view of this it is suggested to screen/select for bacillus spores that are resistant to the inhibitory activity of bile salts, and as a result, germinate into vegetative cells, which then colonize the colon (see [0019]).
The working examples are all in presence and no real experimental data of actually screened specific Bacillus cell are provided in the description.
Further the bile salt screening conditions are relatively generically described. In particular there are no indications of any time periods for the selections of bile resistance. Said in other words, based on the only broad/generic teaching of this document one may select Bacillus cells that only can outgrow (germinate) slowly, i.e. are capable of germinating from spores to vegetative cells after e.g. 20 hours in presence of relevant amount of bile salt.
In this document there is no description or suggestion to select for bacillus cells that can outgrow (germinate) rapidly, i.e. capable of germinating and outgrowing from spores to vegetative cells reaching a defined growth point within a certain time interval in presence of a relevant amount of bile salt.
In summary, the prior art references relating to selection/screening of bile resistant bacillus cells are not focusing on rapid outgrowth/germination from spore cells to vegetative bacillus cells.
The prior art describes a number of tests/screening systems for selection of bacillus strains producing phytase enzymes.
An example is U.S. Pat. No. 6,255,098 in which bacillus strains producing phytase enzymes are identified. Nothing is mentioned about bile resistance of the identified bacillus strains.